Method for stabilizing and selecting recombinant DNA containing host cell

ABSTRACT

The present invention is an improvement in the method for stabilizing and selecting host cells containing recombinant DNA. The method involves transforming a host cell with a recombinant DNA cloning vector which contains the ˜2.5 kb BglII cI repressor gene-containing restriction fragment of bacteriophage λ and then lysogenizing the transformed host cell with a lysogenic organism which contains a marker which is lethal or conditionally lethal in the host cell but which is repressed in the transformed host cell by the repressor gene contained in the recombinant DNA cloning vector. The vector additionally contains a gene which codes for the expression of human proinsulin. The invention further comprises related recombinant DNA cloning vectors, transformed host cells and lysogenized transformed host cells.

CROSS REFERENCE

This application is a continuation-in-part of copending application,Ser. No. 325,511, filed Nov. 27, 1981, now U.S. Pat. No. 4,436,815.

SUMMARY OF THE INVENTION

The invention is an improved selective system that provides a means forstabilizing and selecting recombinant DNA host cells through the use ofa lethal chromosomal marker which is repressed by a gene borne on arecombinant DNA cloning vector. This is particularly important becauserecombinant DNA cloning vectors such as plasmids, are often rapidly lostfrom bacterial populations and industrial scale fermentations mayrequire more than 10¹⁶ cells. Therefore, once the recombinant DNA codingfor the desired product is inserted in a plasmid, it is desirable if notessential, that the microorganism culture containing the plasmid bestabilized so that all the cells comprising the culture will contain thedesired plasmid. This is crucial since recombinant plasmids with foreignDNA are notoriously unstable and often more than 90% of the cells in apopulation may not contain the recombinant plasmid after a culture hasbeen grown overnight. Consequently the productive capacity isdramatically reduced because expression of desired genes is possibleonly in those cells which retain the plasmid.

Very few effective methods have been described for stabilization ofrecombinant plasmids and all have serious disadvantages. One methodinvolves incorporating antibiotic resistance genes into recombinantplasmids and then adding the appropriate antibiotic to the culturemedium. Cells retaining the plasmid with the antibiotic resistance geneare selected for and those which lose the plasmid are selected againstand are therefore eliminated. The major disadvantage of this approach isthat it requires production scale growth of antibiotic resistantbacteria, use of an expensive antibiotic in the fermentation medium, andsubsequent purification to remove the antibiotic from the desiredproduct.

Complementation of an auxotrophic mutation on the chromosome is theother known method for stabilization of recombinant plasmids. Thisapproach severely restricts the composition of the fermentation mediumand requires fermentation in a medium that does not contain the requirednutrient of the host bacteria. Moreover, syntrophism may allow cells tocontinue growth after loss of the plasmid. Therefore, both types ofselection depend on specific manipulation of the media. Suchrestrictions increase the cost of fermentation and limit the optionsavailable for improving productivity.

Alternative selections which are independent of media composition, whichprovide for maintenance of the recombinant DNA cloning vector under allconditions of fermentation, and which allow for enhanced biosynthesis ofa polypeptide product, are urgently needed. Cell suicide is adaptable tosatisfy this need in that suicidal cells containing a lethal marker on achromosome and a repressor or complementing gene on a recombinant DNAcloning vector can be constructed. Cells constructed to thesespecifications will die if they lose the vector. The present inventionimproves this principle by insuring, not only that substantially allviable cells in a culture will carry the desired recombinant cloningvector, but also that the expression of genes contained in the cloningvector is enhanced.

Enhanced expression of product genes and also the absence of plasmidsegregation are particularly advantageous and serve to distinguish thepresent invention from other selective systems which also involve thebacteriophage λcI repressor. Such a selective system, comprising cloningvectors which comprise both the cI repressor gene containing ˜2.5 kbBgIII restriction fragment of bacteriophage λ and also a gene whichexpresses a functional polypeptide, is disclosed in U.S. patentapplication Ser. No. 275,088, filed June 18, 1981, now U.S. Pat. No.4,506,013. Although the gene which codes for the functional polypeptideis expressed, the particular plasmid construction therein disclosedshows some segregation and also does not allow for enhanced and optimumproduction of product. The improved method of the present inventionsolves these problems by affording an excellent means for stabilizingand selecting recombinant DNA containing host cells while concurrentlymaximizing gene expression and biosynthesis of a functional polypeptide.

For purposes of the present invention and as defined herein, arecombinant DNA cloning vector is any agent, including but not limitedto plasmids, bacteriophages, and viruses, consisting of a DNA moleculeto which one or more additional DNA segments can or have been added.

Transformation, as defined herein, is the introduction of DNA into arecipient host cell that changes the genotype and consequently resultsin a heritable change in the recipient cell.

A transformant, as defined herein, is a recipient cell that hasundergone transformation.

A repressor, as defined herein, is a gene which is located on arecombinant DNA coning vector and which represses and preventsexpression of a lethal or conditionally lethal gene in a chromosome of ahost cell.

A functional polypeptide, as defined herein, is a recoverable bioactiveentirely heterologous polypeptide or precursor, a recoverable bioactivepolypeptide comprised of a heterologous polypeptide and a portion orwhole of a homologous polypeptide, a recoverable bioinactive fusionpolypeptide comprised of a heterologous polypeptide and abioinactivating homologous polypeptide which can be specificallycleaved, or a bioactive polypeptide the presence of which can bedetected.

A fused gene product, as defined herein, is a recoverable heterologouspolypeptide which is fused with a portion or whole of a homologouspolypeptide.

A marker, as defined herein, is a gene or combination of genes of knownfunction and location in a chromosome, recombinant DNA cloning vector,or virus.

Ap^(r), as defined herein, designates the ampicillin resistantphenotype.

Ap^(s), as defined herein, designates the ampicillin sensitivephenotype.

Tc^(r), as defined herein, designates the tetracycline resistantphenotype.

Tc^(s), as defined herein, designates the tetracycline sensitivephenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Restriction Site and Functional Map of Plasmid pIA7Δ4Δ1,

FIG. 2--Restriction Site and Functional Map of Plasmid pIB7Δ4Δ1,

FIG. 3--Restriction Site and Functional Map of Plasmid pPR3,

FIG. 4--Restriction Site and Functional Map of Plasmid pPR12,

FIG. 5--Restriction Site and Functional Map of Plasmid pPR17,

FIG. 6--Restriction Site and Functional Map of Plasmid pPR18,

FIG. 7--Thymosin Alpha I Gene,

FIG. 8--Synthesis Procedure for Fragment T₁₅,

FIG. 9--Construction Route for Plasmid pTHα1,

FIG. 10--Proinsulin Gene,

FIG. 11--Restriction Site and Functional Map of Plasmid pPR19.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is an improvement of the method for stabilizingand selecting host cells containing recombinant DNA in which host cellsare transformed with a recombinant DNA cloning vector which containsboth the ˜2.5 kb BglII cI repressor containing restriction fragment ofBacteriophage λ and a gene which expresses a functional polypeptide, andin which the transformed host cells are lysogenized with a lysogenicorganism containing a marker which is lethal or conditionally lethal inthe host cells but which is repressed in the transformed host cell bythe repressor gene contained in the recombinant DNA cloning vector,wherein the improvement comprises transforming the host cells with arecombinant DNA cloning vector comprising:

(a) the PstI-HincII cI repressor containing restriction fragment ofbacteriophage λ; and

(b) a gene which expresses a functional polypeptide;

subject to the limitation that the recombinant DNA cloning vectorcontains a replicon and a promoter which are not sensitive to therepressor, and subject to the further limitation, that when thetransformed host cells are lysogenized with a lysogenic organismcontaining a gene which is conditionally lethal, the resulting hostcells are cultured under restrictive conditions. The invention furthercomprises novel cloning vectors and organisms.

As discussed above, the present invention can be used for the growth ofcultures which produce products coded by recombinant DNA. Without aneffective selective system, many cells in such cultures lose the desiredplasmid and consequently production of the desired product is markedlyreduced. The present invention not only insures that substantially allviable cells in a culture will carry the recombinant DNA cloning vector,but it also enhances gene expression such that greater amounts of afunctional polypeptide are biosynthesized. Therefore, the presentinvention is particularly advantageous and is distinguished by the lackof plasmid segregation, the enhanced level of gene expression, and thesignificantly larger quantities of functional polypeptide produced whenthe improved, as compared to the non-improved, method is in place.

The present invention is particularly versatile since it can be appliedto the production of any substance where synthesis is determined by arecombinant DNA cloning vector. A preferred recombinant DNA cloningvector is the plasmid although bacteriophage and other vectors usefulfor illustrating the present invention will be apparent to those skilledin the art. Since the usefulness of the present invention is independentof the genes that express a functional polypeptide, the invention can beused with recombinant strains that carry one or more genes of commercialvalue. Furthermore, the previously described enhancement of geneexpression is not limited to any particular product gene. Thus, theimproved method of the present invention is advantageous for producingany functional polypeptide or other gene product using recombinant DNAtechniques.

The interaction of bacteriophage λ with E. coli K12 is employed toillustrate the applicability of cell suicide for maintaining andstabilizing recombinant DNA host cells. Bacteriophage λ is a temperatebacteriophage that follows either of two mutually exclusive cycles wheninfecting E. coli K12. In the lytic phase the bacteriophage DNAreplicates autonomously, directs synthesis and assembly of bacteriophagecomponents, and kills the cells concommitant with the release of maturebacteriophage. In the lysogenic phase the bacteriophage is integratedinto the host's chromosome as a prophage, replicates as a marker on thechromosome, and blocks synthesis of bacteriophage components. Abacteriophage gene, λcI, codes for a repressor that maintains thelysogenic state and blocks expression of genes for bacteriophagecomponents and maturation. If the repressor is inactivated or removedfrom the cell, the prophage educts from the chromosome, enters the lyticcycle, and kills the cell. Bacteriophage with a defective λcI genecannot maintain the lysogenic state and are lethal to the cell unless afunctional repressor is provided from an alternate source. In anillustrative embodiment of the present invention, λcI90 is employed as arepressor dependent prophage and a cI gene, contained in a restrictionfragment and cloned into a reoombinant DNA cloning vector, serves as thefunctional repressor.

More particularly, the improved selective system and usefulness of thisinvention can be shown by cloning the plasmid pIA7Δ4Δ1 ˜1.3 kbEcoRI-BamHI restriction fragment, which contains the trpE-insulin Achain gene, onto novel plasmid pPR12. This is done in such a way as todelete the plasmid pPR12˜0.4 kb EcoRI-BamHI segment. Plasmid pPR12 isgenerally useful as a vector since any desirable DNA fragment can beused in place of the plasmid pIA7Δ4Δ1˜1.3 kb restriction fragment.Plasmid pPR12 is constructed by inserting the plasmid pPR3˜0.9 kbPstI-HincII restriction fragment, which contains the bacteriophageλcI857 repressor, onto plasmid pBR322. Plasmid pPR3 is constructed byinserting the 2.5 kb BglII fragment of bacteriophage λcI857 into theunique BamHI restriction site of plasmid pIB7Δ4Δ1. The 2.5 kb BglIIrestriction fragment of bacteriophage λcI857, in addition to containingthe cI repressor gene, also contains the rex gene and part of the crogene. Surprisingly, deletion of the cro gene and most of the rex genefrom the cI repressor gene containing restriction fragment greatlyincreases and enhances genetic expression and thus production offunctional polypeptide. An especially preferred cro and rex deleted λcIcontaining restriction fragment, used herein to exemplify the presentinvention, is the ˜0.9 kb PstI-HincII restriction fragment of plasmidpPR3. A restriction site and functional map of each of plasmidspIA7Δ4Δ1, pIB7Δ4Δ1, pPR3, and pPR12 is presented in FIGS. 1-4 of theaccompanying drawings.

Plasmid pIA7Δ4Δ1, as illustrated herein, contains the E. coli tryptophanpromoter, antibiotic resistance markers, and a gene which expresses afused gene product comprising a portion of the E. coli trp E proteinfused with the A polypeptide chain of human insulin. Plasmid pIB7Δ4Δ1similar except that the gene which expresses the fused gene productcomprises a portion of the trp E protein fused with the B, rather thanthe A, polypeptide chain of human insulin.

Plasmid pIA7Δ4Δ1 is derived from plasmid pBR322 and is constructedaccording to the procedure disclosed in Example 1A-I herein. With regardto conventions, the symbol "Δ" connotes a deletion. Thus, for example,reference to a plasmid followed by, "ΔEcoRI-XbaI" describes the plasmidfrom which the nucleotide sequence between EcoRI and XbaI restrictionenzyme sites has been removed by digestion with those enzymes. Forconvenience, certain deletions are denoted by number. Thus, beginningfrom the first base pair ("bp") of the EcoRI recognition site whichprecedes the gene for tetracycline resistance in the parental plasmidpBR322, "Δ1" connotes deletion of bp 1-30 (ie, ΔEcoRI-HindIII) andconsequent disenabling of the tetracycline promoter/operator system;"Δ2" connotes deletion of bp 1-375 (ie, ΔEcoRI-BamHI) and consequentremoval of both the tetracycline promoter/operator and a portion of thestructural gene which encodes tetracycline resistance; and "Δ4" connotesdeletion of bp ˜900-˜1500 from the trp operon fragment eliminating thestructural gene for the trp D polypeptide.

The cloning of the ˜1.3 kb EcoRI-BamHI trp E-insulin A chain genecontaining restriction fragment of plasmid pIA7Δ4Δ1 onto the ˜4.7 kbEcoRI-BamHI restriction fragment of plasmid pPR12, hereinafterdesignated pPR12Δ2, results in the novel plasmid pPR17. The plasmidpIA7Δ4Δ1 ˜1.3 kb EcoRI-BamHI restriction fragment contains part of Δ2 sotherefore the construction restores Δ2 to Δ1. Plasmid pPR17 contains the˜0.9 kb PstI-HincII restriction fragment of bacteriophage λcI857 andthus blocks the lytic development of bacteriophage lambda in lysogenizedhost cells. In addition, plasmid pPR17 codes for and expresses theaforementioned trp E-insulin A chain fused gene product at levelssignificantly above that of other λcI gene containing plasmids known inthe art. A restriction site and functional map of plasmid pPR17 ispresented in FIG. 5 of the accompanying drawings.

The novel pPR17 recombinant plasmid can be transformed into E. coli suchas, for example, E. coli K12 294 (disclosed in Goeddel et al., 1979,Proc. Nat. Acad. Sci. U.S.A. 76:106), E. coli K12 RV308 (disclosed inMauer et al., 1980, J. Mol. Biol. 139:147-161), E. coli K12 C600(disclosed in Bachman, 1972, Bacteriol. Rev. 36:526-557), E coli K12C600R_(k) -M_(k) -(disclosed in Chang and Cohen, 1974, Proc. Nat. Acad.Sci. 71:1030-1034), and the like, and then the resulting strains can belysogenized with any bacteriophage λ which does not produce a functionalcI repressor such as, for example, bacteriophage λcI90. Thus, theconstructed strains E. coli K12 294λcI90/pPR17, E. coli K12RV308λcI90/pPR17, E. coli K12 C600λcI90/pPR17 and E. coli K12 C600R_(k)-M_(k) -λcI90/pPR17 require retention of the pPR17 plasmid whereasconstructed strains E. coli K12 294/pPR17, E. coli K12 RV308/pPR17, E.coli K12 C600/pPR17, and E. coli K12 C600R_(k) -M_(k) /pPR17 surviveequally well without the plasmid. A comparison of plasmid retention inthe strains clearly demonstrates that substantially all the viable cellsin the strains with the invention have the desired plasmid. Moreover,the E. coli K12 294λcI90/pPR17, E. coli K12 RV308λcI90/pPR17, E. coliK12 C600λcI90/pPR17, and E. coli K12 C600R_(k) -M_(K) -λcI90/pPR17strains not only maintain the pPR17 plasmid, but also produce thedesired fused gene product.

The improved selective system and usefulness of this invention can alsobe shown by cloning the plasmid pIB7Δ4Δ1 ˜1.3 kb EcoRI-BamHI restrictionfragment, which contains the trp E-insulin B chain gene, onto plasmidpPR12 in the manner described for plasmid pPR17. Plasmid pIB7Δ4Δ1 isderived from plasmid pBR322 in a way analogous to that described forpIA7Δ4Δ1. The construction of plasmid pIB7Δ4Δ1 is disclosed in Example 2herein below.

The cloning of the ˜1.3 kb EcoRI-BamHI trp E-insulin B chain genecontaining restriction fragment of plasmid pIB7Δ4Δ1 onto the ˜4.7 kbEcoRI-BamHI restriction fragment of plasmid pPR12 results in the novelplasmid pPR18. Plasmid pPR18 contains the ˜0.9 kb PstI-HincIIrestriction fragment of bacteriophage λcI857 and thus blocks the lyticdevelopment of bacteriophage lambda in lysogenized host cells. Inaddition, plasmid pPR18 codes for and expresses the aforementioned trpE-insulin B chain fused gene product at levels significantly above thatof other λcI gene containing plasmids known in the art. A restrictionsite and functional map of plasmid pPR18 is presented in FIG. 6 of theaccompanying drawings.

The novel pPR18 recombinant plasmid can be transformed into E. coli suchas, for example, E. coli K12 294, E. coli K12 RV308, E. coli K12 C600,E. coli K12 C600R_(k) -M_(k) - and the like, and then the resultingstrains can be lysogenized with any bacteriophage λ which does notproduce a functional cI repressor such as, for example, bacteriophageλcI90. Thus, as was previously described for the lysogenized pPR17containing strains, the constructed E. coli K12 294λcI90/pPR18, E. coliK12 RV308λcI90/pPR18, E. coli K12 C600λcI90/pPR18, and E. coli K12 C600R_(k) -M_(k) -λcI90/pPR18 strains require retention of the pPR18 plasmidwhereas constructed strains E. coli K12 294/pPR18, E. coli K12RV308/pPR18, E. coli K12 C600/pPR18, and E. coli K12 C600R_(k) -M_(k)-/pPR18 do not and survive equally well without the plasmid. Acomparison of plasmid retention in the strains clearly demonstrates thatsubstantially all the viable cells in the strains with the inventionhave the desired plasmid. Moreover, the E. coli K12 294λcI90/pPR18, E.coli K12 RV308λcI90/pPR18, E. coli K12 C600λcI90/pPR18, and E. coli K12C600-λcI90/pPR18, and E. coli K12 C600R_(k) -M_(k) -λcI90/pPR18 strainsnot only maintain their plasmids but also produce the desired fused geneproduct.

Novel plasmid pPR12 can also be transformed into a variety of hostcells, such as, for example, E. coli K12 C600R_(k) -M_(k) -. Theresulting strains can be lysogenized, as in the case of the plasmidpPR17 and pPR18 transformants, to produce strains that do not survivewithout the plasmid. Thus, constructed strain E. coli K12 C600R_(k)-M_(k) -λcI90/pPR12 requires retention of the pPR12 plasmid whereasconstructed strain E. coli K12 C600R_(k) -M_(k) -/pPR12 survives equallywell without the plasmid. A comparison of plasmid retention in thestrains clearly demonstrates that substantially all the viable cells inthe strain with the invention have the desired plasmid.

The λcI857 repressor gene used herein to illustrate the presentinvention is temperature sensitive and is inactivated at 38° C. to 44°C. or above. A temperature shift from 38° C. to 44° C. therefore lysesthe cells by inducing the lytic cycle of the lambda prophage which, inaccordance with the present invention, has been incorporated into thehost cell strain. As is readily apparent, when a temperature sensitiverepressor which represses a lethal or conditional lethal marker thatcauses host cell lysis is used and when the host cells are cultured at atemperature which inactivates the repressor and, in the case of aconditional lethal marker, at a temperature which is not within thetemperature range for permissive culture of the host cells, the improvedselection method of the present invention also provides a simple,convenient, and inexpensive procedure to lyse cells for purification ofintracellular products.

As illustrated herein, the present invention employs a plasmid bornegene to repress a lethal chromosomal marker. Selection of cells isindependent of the replicon and also the other genes on the plasmid and,although the preferred embodiment herein described employs the ˜0.9 kbPstI-HincII cI containing restriction fragment of bacteriophage λcI857,the ˜0.9 kb PstI-HincII cI containing restriction fragment of any otherbacteriophage λ strain that produces a functional repressor can also beused. Furthermore, although the prophage used to exemplify the presentinvention carries a λcI90 mutation and thus does not produce afunctional λcI repressor, other bacteriophage λ mutants can also beemployed if they too lack a functional cI repressor gene. As is readilyapparent, such mutants require an alternate source of repressor tomaintain the lysogenic state.

The selective method of the present invention allows for the enhancedexpression of a functional polypeptide and can be imposed on host cellscontaining plasmid borne genes that express a variety of usefulproducts. For example, the plasmid borne gene may be a naturallyoccurring gene, non-naturally occurring gene, or a gene which is in partnaturally occurring and in part synthetic or non-naturally occurring.More particularly, the invention can be used to select and maintaincells containing a plasmid borne gene coding for human pre-proinsulin,human proinsulin, human insulin A-chain, human insulin B-chain, humangrowth hormone, non-human growth hormone, nonhuman insulin, humaninterferon, nonhuman interferon, viral antigen, urokinase, any peptidehormone, any enzyme, any polypeptide, or for virtually any other genewith research or commercial value.

In the specific embodiments of the invention described herein, plasmidreplication and expression of the gene product are determinedrespectively by the replicon from pMBl (disclosed in Bolivar, 1979, LifeSci. 25:807-818) and by the trp promoter. Other replicons and promoterscan also be used so long as they are functional in E. coli K12 and arenot sensitive to the particular repressor being used. It is understoodthat those skilled in the art know or readily can determine whichreplicons and promoters are functional in E. coli K12 and whioh are notsensitive to a particular repressor. Examples of other replicons includebut are not limited to replicons from ColEl, NRl, RK2, RK6, pSC101, RP1,RP4, F, and the like, including bacteriophage that replicate in E. coliK12. Examples of other promoters include but are not limited to the lacpromoter, lipoprotein promoter, ribosomal protein or RNA promoters, andvirtually any other promoter. It is understood that other replicons andpromoters can be constructed and will be apparent to those skilled inthe art.

In addition to being the preferred host for bacteriophage λ, the wealthof genetic and biochemical information about E. coli K12 makes it aconvenient host cell for purposes of the present invention. Although thestrain E. coli K12 RV308 is most preferred, the invention is not limitedto any one genus, species, or strain, but can be used with any E. coli,coliform, or other cell in which bacteriophage λ is lysogenic and intowhich a functional repressor can be cloned.

All of the embodiments of the present invention share the common featurethat they are insensitive to media composition. Therefore, the inventionallows for a wide range of fermentation manipulation to improveproductivity.

The following examples further illustrate and also present a preferredembodiment of the invention disclosed herein. Both an explanation of andthe actual procedures for constructing the invention are described whereappropriate.

EXAMPLE 1 Construction of Plasmid pIA7Δ4Δ1

A. Construction of Plasmid pBRHtrp

Plasmid pGM1 carries the E. coli tryptophan operon containing thedeletion ΔLE1413 (Miozzari, et al., 1978, J. Bacteriology, 1457-1466)and hence expresses a fusion protein comprising the first 6 amino acidsof the trp leader and approximately the last third of the trp Epolypeptide (hereinafter referred to in conjunction as LE'), as well asthe trp D polypeptide in its entirety, all under the control of the trppromoter-operator system. E. coli K12 W3110tna2trpΔ102/pGM1 has beendeposited with the American Type Culture Collection (ATCC No. 31622) andpGM1 may be conventionally removed from the strain for use in theprocedures described below.

About 20 μg of the plasmid were digested with the restriction enzyme*PvuII which cleaves the plasmid at five sites. The gene fragments werenext combined with EcoRI linkers (consisting of a self complementaryoligonucleotide of the sequence: pCATGAATTCATG) providing an EcoRIcleavage site for later cloning into a plasmid containing an EcoRI site.The 20 μg of DNA fragments obtained from pGM1 were treated with 10 unitsT₄ DNA ligase in the presence of 200 pico moles of the 5'-phosphorylatedsynthetic oligonucleotide pCATGAATTCATG and in 20 μl T₄ DNA ligasebuffer (20 mM tris, pH 7.6, 0.5 mM ATP, 10 mM MgCl₂, 5 mMdithiothreitol) at 4° C. overnight. The solution was then heated 10minutes at 70° C. to halt ligation. The linkers were cleaved by EcoRIdigestion and the fragments, now with EcoRI ends, were separated using 5percent polyacrylamide gel electrophoresis (herein after "PAGE"). Thethree largest fragments were isolated from the gel by first stainingwith ethidium bromide and then locating the fragments with ultravioletlight and cutting from the gel the portions of interest. Each gelfragment, with 300 microliters 0.1×TBE, was placed in a dialysis bag andsubjected to electrophoresis at 100 v for one hour in 0.1×TBE buffer(TBE buffer contains: 10.8 gm tris base, 5.5 gm boric acid, 0.09 gm Na₂EDTA in 1 liter H₂ O). The aqueous solution was collected from thedialysis bag, phenol extracted, chloroform extracted, and made 0.2M withrespect to sodium chloride. The DNA was then recovered in water afterethanol precipitation. The trp promoter/operator-containing gene withEcoRI sticky ends was identified in the procedure next described, whichentails the insertion of fragments into a tetracycline sensitive plasmidwhich, upon promoter/operator insertion, becomes tetracycline resistant.All DNA fragment isolations hereinafter described are performed usingPAGE followed by the electroelution method described above.

B. Construction of Plasmid pBRH trp Expressing Tetracycline ResistanceUnder the Control of the Trp Promoter/Operator and Identification andAmplification of the Trp Promoter/Operator Containing DNA FragmentIsolated in `A` above

Plasmid pBRH1, (Rodriguez, et al., 1979, Nucleic Acids Research 6,3267-3287) expresses ampicillin resistance and contains the gene fortetracycline resistance but, there being no associated promoter, doesnot express that resistance. The plasmid is accordingly tetracycinesensitive. By introducing a promoter/operator system in the EcoRI site,the plasmid can be made tetracycline resistant.

Plasmid pBRH1 was digested with EcoRI. The enzyme was removed by phenolextraction followed by chloroform extraction and then the DNA wasrecovered in water after ethanol precipitation. The resulting DNAmolecule was, in separate reaction mixtures, combined with each of thethree DNA fragments obtained in Example 1A above and ligated with T₄ DNAligase as previously described. The DNA present in the reaction mixturewas used to transform competent E. coli K12 strain 294, (Backman et al.,1976, Proc. Nat. Acad. Sci. U.S.A 73:4174-4198, ATCC No. 31446) bystandard techniques (Hershfield et al., 1974, Proc. Nat. Acad. Sci.U.S.A 71:3455-3459) and the bacteria were then plated on LB plates(Miller, 1972) containing 20 μg/ml ampicillin and 5 μg/ml tetracycline.

Several tetracycline-resistant colonies were selected and the plasmidDNA was isolated and designated pBRHtrp. The presence of the desiredfragment was confirmed by restriction enzyme analysis. Plasmid pBRH trpexpresses β-lactamase, imparting ampicillin resistance, and contains aDNA fragment which includes the trp promoter/operator. The DNA fragmentalso codes for a first protein, (designated LE'), comprising a fusion ofthe first six amino acids of the trp leader and approximately the lastthird of the trp E polypeptide, a second protein (designated D'),corresponding to approximately the first half of the trp D polypeptide,and a third protein, coded for by the tetracycline resistance gene.

C. Construction of Plasmid pSOM7Δ2

Plasmid pBRHtrp was digested with EcoRI restriction enzyme and theresulting fragment, isolated by PAGE and electroelution, was combinedwith EcoRI-digested plasmid pSOM11 (Itakura et al., 1977, Sci. 198:1056,G. B. Patent Publication No. 2,007,676A). The mixture was ligated withT₄ DNA ligase and the resulting DNA transformed into E. coli K12 strain294 as previously described. Transformant bacteria were selected onampicillin-containing plates and the resulting ampicillin-resistantcolonies were screened by colony hybridization (Gruenstein et al., 1975,Proc. Nat. Acad. Sci. U.S.A. 72:3951-3965). The trppromoter/operator-containing fragment, isolated from pBRH trp and thenradioactively labelled with p³², was used as a probe in the aboveprocedure. Several colonies were shown to be positive by colonyhybridization and were therefore selected. Plasmid DNA was isolated andthe orientation of the inserted fragments was determined by restrictionanalysis, using enzymes BglII and BamHI in double digestion. Coloniescontaining the desired plasmid with the trp promoter/operator fragmentin the proper orientation were grown in LB medium (Miller, 1972)containing 10 μg/ml ampicillin. The desired plasmid was designatedpSOM7Δ2 and was used for subsequent constructions described below.

D. Construction of Plasmid pTrp24

1. Construction of a Gene Fragment Comprisinq Codons for the DistalRegions of the LE' Polypeptide With BglII and EcoRI Restriction SitesRespectively at the 5' and 3' Ends of the Coding Strand

Plasmid pSOM7Δ2 was HindIII digested followed by digestion with lambdaexonuclease (a 5' to 3' exonuclease) under conditions chosen so as todigest beyond the BglII restriction site within the LE' encoding region.About 20 μg of HindIII-digested pSOM7Δ2 was dissolved in buffer (20 mMglycine buffer, pH 9.6, 1 mM MgCl₂, 1 mM β-mercaptoethanol). Theresulting mixture was treated with 5 units of lambda exonuclease for 60minutes at room temperature. The reaction mixture obtained was thenphenol extracted, chloroform extracted, and ethanol precipitated.

To create an EcoRI residue at the distal end of the LE' gene fragment, aprimer ³² pCCTGTGCATGAT was synthesized by the improved phosphotriestermethod (Crea et al., 1978, Proc. Nat. Acad. Sci. U.S.A. 75:5765) andhybridized to the single stranded end of the LE' gene fragment resultingfrom lambda exonuclease digestion. The hybridization was performed bydissolving 20 μg of the lambda exonuclease-treated HindIII digestionproduct of plasmid pSOM7Δ2 in 20 μl H₂ O and combining with 6 μl of asolution containing approximately 80 picomoles of the 5'-phosphorylatedoligonucleotide described above. The synthetic fragment was hybridizedto the 3' end of the LE' coding sequence and the remaining single strandportion of the LE' fragment was filled in by Klenow Poymerase I usingdATP, dTTP, dGTP and dCTP. Klenow Polymerase I is the fragment obtainedby proteolytic cleavage of DNA Polymerase I. It contains the 5'→3'polymerizing activity, the 3'→5' exonucleolytic activity, but not the5'→3' exonucleolytic activity of the parental enzyme (Kornberg, 1974, W.H. Freeman and Co., SFO, 98).

The reaction mixture was thus heated to 50° C. and let cool slowly to10° C., whereafter 4 μl of Klenow enzyme were added. After 15 minutesincubation at room temperature, followed by 30 minutes incubation at 37°C., the reaction was stopped by the addition of 5 μl of 0.25 molar EDTA.The reaction mixture was phenol extracted, chloroform extracted, andethanol precipitated. The DNA was subsequently cleaved with therestriction enzyme BglII and the fragments were separated by PAGE. Anautoradiogram obtained from the gel revealed a ³² P-labelled fragment ofthe expected length of approximately 470 bp, which was recovered byelectroelution. As outlined, this fragment LE'(d) has a BglII terminusand a blunt end coinciding with the beginning of the primer.

2. Construction of Plasmid pThα1

Plasmid pThα1 was constructed by inserting a synthesized gene forthymosin alpha 1 into plasmid pBR322. The synthesis of the thymosinalpha 1 coding DNA involves the synthesis and subsequent ligation of the16 oligonucleotides (T₁ through T₁₆) that are indicated by the doubleheaded arrows in FIG. 7 of the accompanying drawings. A Met codon ATGwas inserted at the N-terminus and the 5' ends were designed withsingle-stranded cohesive termini to facilitate joining to plasmidscleaved with EcoR1 and BamH1. As can be readily appreciated, the BglIIsite in the center of the gene assists in the analysis of recombinantplasmids.

Oligodeoxyribonucleotides T₁ to T₁₆ were synthesized by the modifiedphosphotriester method using fully protected trideoxyribonucleotidebuilding blocks (Itakura et al., 1977, Science 198:1056, and Crea etal., 1978). The various oligodeoxyribonucleotides are shown below inTable 1.

                                      TABLE 1                                     __________________________________________________________________________    SYNTHETIC OLIGONUCLEOTIDES FOR THYMOSINα1 GENE                                                               HPLC Analysis                                                                 Retention                                Compound                                                                            Sequence                   Length                                                                            Time (min)*                              __________________________________________________________________________    T.sub.1                                                                             A--A--T--T--C--A--T--G--T--C                                                                             10  17.4                                     T.sub.2                                                                             T--G--A--T--G--C--T--G--C--T--G--T--T--G--A                                                              15  24.3                                     T.sub.3                                                                             T--A--C--T--T--C--T--T--C--T--G--A                                                                       12  20.3                                     T.sub.4                                                                             G--A--T--T--A--C--T--A--C--T--A--A--A                                                                    13  22.0                                     T.sub.5                                                                             G--C--A--G--C--A--T--C--A--G--A--C--A--T--G                                                              15  24.8                                     T.sub.6                                                                             G--A--A--G--T--A--T--C--A--A--C--A                                                                       12  20.1                                     T.sub.7                                                                             A--G--T--A--A--T--C--T--C--A--G--A--A                                                                    13  22.6                                     T.sub.8                                                                             A--A--G--A--T--C--T--T--T--A--G--T                                                                       12  20.2                                     T.sub.9                                                                             G--A--T--C--T--T--A--A--G--G--A--G                                                                       12  20.4                                     T.sub.10                                                                            A--A--G--A--A--G--G--A--A--G--T--T                                                                       12  21.1                                     T.sub.11                                                                            G--T--C--G--A--A--G--A--G--G--C--T                                                                       12  20.5                                     T.sub.12                                                                            G--A--G--A--A--C--T--A--A--T--A--G                                                                       12  20.4                                     T.sub.13                                                                            C--T--T--C--T--T--C--T--C--C--T--T                                                                       12  19.9                                     T.sub.14                                                                            T--T--C--G--A--C--A--A--C--T--T--C                                                                       12  20.5                                     T.sub.15                                                                            G--T--T--C--T--C--A--G--C--C--T--C                                                                       12  20.2                                     T.sub.16                                                                            G--A--T--C--C--T--A--T--T--A                                                                             10  17.2                                     __________________________________________________________________________     *at ambient temperature                                                  

The above synthesis is typified by the following procedure for fragmentT₁₅ as summarized in FIG. 8 of the accompanying drawings. Variousnucleotide fragments that are used in the synthesis of T₁₅ arenumerically designated in the Figure. The abbreviations employed are asfollows: TPSTe, 2,4,6-triisopropylbenzenesulfonyltetrazole; BSA, benzenesulfonic acid; TLC, thin layer chromatography; HPLC, high performanceliquid chromatography; DMT, 4,4'-dimethoxytrityl; CE, 2-cyanoethyl; R,p-chlorophenyl; Bz, benzoyl; An, anisoyl; iBu, isobutryl; Py, pyridine;AcOH, acetic acid; Et₃ N, triethylamine.

The fully protected trideoxyribonucleotides 4 (85 mg, 0.05 mmol) and 2(180 mg, 0.1 mmol) were deblocked at the 5' hydroxyls by treatment with2% BSA in 7:3 (v/v) chloroform/methanol (10 and 20 ml, respectively) for10 minutes at 0° C. Reactions were stopped by addition of saturatedaqueous ammonium bicarbonate (2 ml), extracted with chloroform (25 ml),and washed with water (2×10 ml). The organic layers were dried(magnesium sulfate), concentrated to small volumes (about 5 ml), andprecipitated by addition of petroleum ether (35°-60° C. fraction). Thecolorless precipitates were collected by centrifugation and dried in adessicator in vacuo to give 6 and 8, respectively, each homogeneous bysilica gel tlc (Merck 60 F254, chloroform/methanol, 9:1).

Trimers 1 and 3 (270 mg, 0.15 mmol; 145 mg, 0.075 mmol) were convertedinto their phosphodiesters (5 and 7) by treatment withtriethylamine/pyridine/water (1:3:1, v/v, 10 ml) for 25 minutes atambient temperature. Reagents were removed by rotary evaporation and theresidues dried by repeated evaporations with anhydrous pyridine (3×10ml). Trimer 8 (0.05 mmol) and trimer 7 were combined with TPSTe (50 mg,0.15 mmol) in anhydrous pyridine (3 ml) and the reaction mixture left invacuo at ambient temperature for two hours. TLC analysis showed that 95%of the trimer 8 had been converted into hexamer product (visualized bydetection of the DMT group by spraying with 10% aqueous sulfuric acidand heating at 60° C.). The reaction was quenched by addition of water(1.0 ml) and the solvent evaporated under reduced pressure. Afterremoval of pyridine by coevaporations with toluene, the hexamer wasdeblocked at the 5' position with 2% BSA (8 ml) as described above fortrimers 4 and 2. The product (10) was purified on a silica gel column(Merck 60 H, 3.5×5 cm) by step gradient elution with chloroform/methanol(98:2 to 95:5, v/v). Fractions containing product 10 were evaporated todryness.

Similarly, trimer 5 was coupled to 6 and the fully protected productdirectly purified on silica gel. This latter compound was deblocked atthe 3' end by triethylamine/pyridine/water as described above to givefragment 9.

Finally, hexamers 9 and 10 were coupled in anhydrous pyridine (2 ml)with TPSTe (75 mg, 0.225 mmol) as the condensing agent. Upon completion(4 hours, ambient temperature) the mixture was rotary evaporated and theresidue chromatographed on silica gel. Product 11 (160 mg) was obtainedby precipitation with petroleum ether and appeared homogeneous on TLC. Aportion of compound 11 (20 mg) in pyridine (0.5 ml) was completelydeblocked by treatment with concentrated ammonium hydroxide (7 ml, 8hours, 60° C.) and subsequent treatment in 80% acetic acid (15 minutes,ambient temperature). After evaporation of acetic acid, the solidresidue was dissolved in 4% aqueous ammonium hydroxide (v/v, 4 ml) andextracted with ethyl ether (3×2 ml). The aqueous phase was concentratedto 1-2 ml and a portion applied to HPLC for purification of 12. Thefractions corresponding to the major peak were pooled (ca. 2.0 O. D.₂₅₄units) and concentrated to about 5 ml. The final product 12 was desaltedon Bio-gel P-2 (1.5×100 cm) by elution with 20% aqueous ethanol, reducedto dryness and resuspended in water (200 μl) to give a solution of A₂₅₄=10. The sequence of 12 was confirmed by two-dimensional sequenceanalysis.

The complete thymosin alpha 1 gene was assembled from the 16 syntheticoligo-nucleotides by methods previously described in detail forsomatostatin (Itakura et al., 1977), insulin (Goeddel et al., 1979), andgrowth hormone (Goeddel, Heyneker, et al., 1979, Nature 281:544). Tenmicrogram quantities of oligonucleotides T₂ through T₁₅ werequantitatively phosphorylated with [γ-³² P]-ATP (New England Nuolear) inthe presence of T₄ polynucleotide kinase (Goeddel et al, 1979), to givespecific activities of approximately 1 Ci/mmol. Radio-labelled fragmentswere purified by 20% polyacrylamide/7 M urea gel electrophoresis andsequences of the eluted fragments were verified by two-dimensionalelectrophoresis/homochromatography (Jay et al., 1974, Nucleic Acids Res.1:331) of partial snake venom digests. Fragments T₁ and T₁₆ were leftunphosphorylated to minimize undesired polymerization during subsequentligation reactions. These oligonucleotides (2 μg each) were assembled infour groups of four fragments (see FIG. 9 of the accompanying drawings),by T₄ DNA ligase using published procedures (Goeddel et al., 1979). Thereaction products were purified by gel electrophoresis on a 15%polyacrylamide gel containing 7 M urea (Maxam and Gilbert, 1977, Proc.Nat. Acad. Sci. U.S.A. 71:3455). The four isolated products were ligatedtogether and the reaction mixture resolved by 10% polyacrylamide gelelectrophoresis. DNA in the size range of the thymosin alpha 1 gene(90-105 base pairs) was electroeluted.

Plasmid pBR322 (0.5 μg) was treated with BamHI and EcoRI restrictionendonucleases and the fragments separated by polyacrylamide gelelectrophoresis. The large fragment was recovered from the gel byelectroelution and subsequently ligated to the assembled synthetic DNA(Goeddel, Heyneker, et al., 1979). This mixture was used to transform E.coli K12 strain 294, ATCC No. 31446. Five percent of the transformationmixture was plated on LB plates containing 20 μg/ml ampicillin. The fourampicillin resistant colonies obtained were sensitive to tetracycline,suggesting insertion into the tetracycline resistance gene. Analysis ofthe plasmids from these four colonies showed that in each case theplasmid, designated pThα1, contained (a) a BglII site not found inpBR322 itself, thus indicating the presence of the thymosin alpha 1 geneas shown in FIG. 7, and (b) a fragment of approximately 105 base pairsgenerated by BamHI/EcoRI cleavage. The construction route for plasmidpThα1 (not drawn to scale), is presented in FIG. 9 of the accompanyingdrawings wherein the heavy dots indicate 5'-phosphate groups.

3. Reaction of Treated pThα1 and LE'(d) Fragment

The plasmid pThα1 contains a gene specifying ampicillin resistance and astructural gene specifying thymosin alpha 1 cloned at its 5' codingstrand end into an EcoRI site and at its 3' end into a BamHI site. Thethymosin qene contains a BglII site as well. To create a plasmid capableof accepting the LE'(d) fragment prepared above, pThα1 was EcoRIdigested followed by Klenow polymerase I reaction with dTTP and dATP toblunt the EcoRI residues. BglII digestion of the resulting productcreated a linear DNA fragment containing the gene for ampicillinresistance and, at its opposite ends, a sticky BglII residue and a bluntend. The resulting product could be recircularized by reaction with theLE'(d) fragment containing a BglII sticky end and a blunt end in thepresence of T₄ ligase to form the plasmid pTrp24. In doing so, an EcoRIsite is recreated at the position where blunt end ligation occurred.

E. Construction of Plasmid pSOM7Δ2Δ4

Successive digestion of pTrp24 with BglII and EcoRI, followed by PAGEand electroelution, yields a fragment having codons for the LE'(d)polypeptide with a BglII sticky end and an EcoRI sticky end adjacent toits 3' coding terminus. The LE'(d) fragment can be cloned into the BglIIsite of plasmid pSOM7Δ2 to form an LE' polypeptide/somatostatin fusionprotein expressed under the control of the tryptophan promoter/operator.To do so requires (1) partial EcoRI digestion of pSom7Δ2 in order tocleave the EcoRI site distal to the tryptophan promoter/operator, and(2) proper choice of the primer sequence in order to properly maintainthe codon reading frame, and to recreate an EcoRI cleavage site.

Thus, 16 μg of plasmid pSOM7Δ2 was diluted into 200 μl of buffercontaining 20 mM Tris, pH 7.5, 5 mM MgCl₂, 0.02 NP40 detergent, and 100mM NaCl, and treated with 0.5 units EcoRI. After 15 minutes at 37° C.,the reaction mixture was phenol extracted, chloroform extracted, ethanolprecipitated, and subsequently digested with BglII. The larger resultingfragment was isolated by the PAGE procedure followed by electroelution.This fragment contains the codons "LE'(p)" for the proximal end of theLE' polypeptide, i.e., those upstream from the BglII site. This fragmentwas next ligated to the above LE'(d) fragment in the presence of T₄ DNAligase to form the plasmid pSOM7Δ2Δ4, which upon transformation into E.coli strain 294, efficiently produced a fusion protein consisting of thefully reconstituted LE polypeptide and somatostatin under the control ofthe tryptophan promoter/operator.

F. Construction of Linear DNA Having a PstI Residue at the 3' end and aBglII Residue at its 5' End Bounding a Gene Specifying TetracyclineResistance

Plasmid pBR322 was HindIII digested and the protruding HindIII ends weredigested with S1 nuclease. The S1 nuclease digestion involved treatmentof 10 μg of HindIII-cleaved pBR322 in 30 μl S1 buffer (0.3M NaCl 1 mMZnCl₂, 25 mM sodium acetate, pH 4.5) with 300 units S1 nuclease for 30minutes at 15° C. The reaction was stopped by the addition of 1 μl of 30X S1 nuclease stop solution (0.8M tris base, 50 mM EDTA). The mixturewas phenol extracted, chloroform extracted, ethanol precipitated, andthen EcoRI digested as previously described. The resulting fragment,obtained by the PAGE procedure followed by electroelution, has an EcoRIsticky end and a blunt end whose coding strand begins with thenucleotide thymidine. The S1-digested HindIII residue beginning withthymidine can be joined to a Klenow Polymerase I-treated BglII residueso as to reconstitute the BglII restriction site upon ligation.

Therefore plasmid pSOM7Δ2, prepared in Example 1C, was BglII digestedand the resulting BglII sticky ends were made double stranded bytreatment with Klenow Polymerase I using all four deoxynucleotidetriphosphates. EcoRI cleavage of the resulting product, followed by PAGEand electroelution of the small fragment, yielded a linear piece of DNAcontaining the tryptophan promoter/operator and codons of the LE'"proximal" sequence upstream from the BglII site ("LE'(p)"). The producthad an EcoRI end and a blunt end resulting from filling in the BglIIsite. However, the BglII site is reconstituted by ligation of the bluntend to the blunt end of the above S1-digested HindIII fragment. Thus,the two fragments were ligated in the presence of T₄ DNA ligase to formthe recircularized plasmid pHKY10 which was propagated by transformationinto competent E. coli strain 294 cells. Tetracycline resistant cellsbearing the recombinant plasmid pHKY10 were selected and the plasmid DNAextracted. Digestion with BglII and PstI, followed by isolation by thePAGE procedure and electroelution of the large fragment, yielded thedesired linear piece of DNA having PstI and BglII sticky ends. This DNAfragment, thus produced from pHKY10, contains the origin of replicationand therefore is useful as a component in the construction. of plasmidpIA7Δ4Δ1 in which both the genes coding for the trp LE' polypeptidefusion protein and the tetracycline resistance are controlled by the trppromoter/operator.

G. Construction of Linear DNA Having the Trp Promoter/Operator

Plasmid pSOM7Δ2Δ4, prepared in Example 1E, was subjected to partialEcoRI digestion followed by PstI digestion. The resulting fragmentcontained the trp promoter/operator and was isolated by the PAGEprocedure followed by electroelution. Partial EcoRI digestion wasnecessary to obtain a fragment which was cleaved adjacent to the 5' endof the somatostatin gene but not cleaved at the EcoRI site presentbetween the ampicillin resistance gene and the trp promoter/operator.Ampicillin resistance lost by the PstI cut in the ampicillin resistancegene can be restored upon ligation with the final pHKY10 linear DNAderivative produced in Example 1F above.

H. Isolation of the Insulin A Chain Structural Gene

The insulin A chain structural gene was obtained by the EcoRI and BamHIdigestion of plasmid pIA1, whose construction is disclosed in Goeddel etal., 1979, Proc. Nat. Acad. Sci. U.S.A. 76:106. The desired fragment waspurified by PAGE and electroelution and had EcoRI and BamHI termini.

I. Ligation of the Insulin A Chain Structural Gene, the TrpPromoter/Operator, and the pHKY10 Linear DNA Fragment Having PstI andBglII Termini

The insulin A chain structural gene, the linear DNA fragment containingthe trp promoter/operator (prepared in Example 1G), and the pHKY10linear DNA fragment (prepared in Example 1F), were ligated together inproper orientation, as depicted in FIG. 1, to form the desired plasmidpIA7Δ4Δ1. Plasmid pIA7Δ4Δ1 can be readily selected because of therestoration of ampicillin and tetracycline resistance.

EXAMPLE 2 Construction of Plasmid pIB7Δ4Δ1

The desired plasmid was constructed in accordance with Example 1A-Iexcept that the structural gene specifying the insulin B chain, ratherthan the insulin A chain, was used in the final ligation. The insulin Bchain structural gene wa obtained by the EcoRI and BamHI digestion ofplasmid pIB1, whose construction is disclosed in Goeddel et. al., 1979.The insulin B chain encoding DNA fragment was purified by PAGE andelectroelution and had EcoRI and BamHI termini.

Plasmid pIB7Δ4Δ1 is depicted in FIG. 2 and can be readily selectedbecause of the restoration of ampicillin and tetracycline resistance.

EXAMPLE 3 Construction of Plasmid pPR3

A. Isolation of a ˜2.5 kb BglII Restriction Fragment of Bacteriophage λContaining Genes For cI, rex, and Part of cro

The several BgIII restriction sites in bacteriophage λcI857 and a singleBamHI restriction site in plasmid pIB7Δ4Δ1 allow for the cloning ofbacteriophage fragments into the pIB7Δ4Δ1 cloning vector. BacteriophageλcI857 contains six sites that are sensitive to BglII. One of the BglIIfragments contains 2.5 kb including the λcI gene and also the λrex gene(Szybalski and Szybalski, 1979, Gene 7:217-280 and O'Brien, ed., March1980, Genetic Maps, Vol. 1, NIH). BglII fragments contain 5' extensionswith the sequence GATC that are complementary to 5' extensions on BamHIfragments. Human insulin plasmid pIB7Δ4Δ1 contains a single site that iscleaved by BamHI. Cloning into the BamHI site inactivates the Tcresistance gene carried on pIB7Δ4Δ1. Ligation of BglII fragments andBamHI fragments produces recombinants with the sequences ##STR1## at thejunctions. These sequences are not cleaved by BglII or BamHI. Therefore,restriction with both enzymes eliminates all ligation products exceptthose containing a λBglII fragment ligated into the BamHI site ofpIB7Δ4Δ1.

Restriction enzymes were purchased from commercial sources, disclosed inExample 1A, and were conventionally used according to known procedures.In addition, instructions are also supplied by the manufacturers. Thus,bacteriophage λcI₈₅₇ sus S₇ DNA was restricted to completion at 37° C.in a reaction mixture containing 100 μg of DNA, 12 mM Tris.HCl, pH 7.5,12 mM MgCl₂, 12 mM 2-mercaptoethanol and 100 units (in a volume of 1 ml)of BglII restriction enzyme. The restriction fragments were separated byagarose gel electrophoresis (hereinafter "AGE"). The separated fragmentswere located in the gel by staining with ethidium bromide andvisualizing fluorescent bands with an ultraviolet light. The ˜2.5 kbfragment of interest was excised from the gel and electroeluted into TBEas taught in Example 1A. The aqueous solution was collected from thedialysis bag and passed over a DEAE Cellulose column *(0.5 ml WhatmanDE52) that had been equilibrated with equilibration buffer (0.1 M KCl,10.0 mM Tris.HCl, pH 7.8). The column was washed with 2.5 ml ofequilibration buffer and the DNA (about 5 μg) was eluted with 1.5 ml ofelution buffer (1 M NaCl, 10 mM Tris.HCl, pH 7.8). The eluent wasadjusted to about 0.35 M with respect to Na⁺ ion concentration, and thenthe DNA was precipitated by addition 2 volumes (about 9 ml) of 100%ethanol followed by cooling to -20° C. for 16 hr. The DNA precipitatewas pelleted by centrifugation, washed with 75% ethanol, and dried. TheDNA fragment isolations were performed by the AGE, electroelution,DEAE-Cellulose chromatography, and the ethanol precipitation procedureherein described. The DNA was redissolved in TE buffer (1 mM EDTA, 10 mMTris.HCl, pH 7.8).

B. Digestion of Plasmid pIB7Δ4Δ1 with BamHI Restriction Enzyme

Plasmid pIB7×4Δ1 was restricted to completion at 37° C. in a 50 μlreaction mixture containing 20 mM Tris.HCl, pH 7.0, 100 mM NaCl, 7 mMMgCl₂, 2 mM 2-mercaptoethanol, and 10 units of BamHI restrictionendonuclease.

C. Ligation of the λcI Fragment with BglII Termini to the BamHI DigestedpIB7×4Δ1

Ligation with T4 DNA ligase was performed in a 100 μl reaction mixturecontaining about 1.4 μg of the 2.5 kb BglII fragment (prepared inExample 3A), about 1.5 μg of BamHI restricted pIB7Δ4Δ1 (prepared inExample 3B), 50 mM Tris.HCl, pH 7.8, 10 mM dithiothreitol, 5% glycerol,10 mM MgCl₂, 0.1 mM ATP, and 0.1 unit of T4 DNA ligase. The reactionmixture was incubated at 4° C. for 18 hrs and then terminated by heatingto 65° C. for 5 minutes. The thus prepared plasmid pPR3 was stored at 4°C. for future use.

EXAMPLE 4 Transformation of Plasmid PR3 Into E. coli K12 C600R_(K)-M_(K) -

Fresh overnight cultures of E. coli K12 C600R_(K) M_(K) - (disclosed inChang and Cohen, 1974, Proc. Nat. Acad. Sci. 71:1030-1034) weresubcultured 1:10 in fresh L-broth (disclosed in Miller, 1972,Experiments in Molecular Genetics, Cold Spring Harbor Labs, Cold SpringHarbor, N.Y.) and grown at 37° C. for 1 hr. A total of 660 Klett unitsof cells were harvested, washed with 2.5 ml of 100 mM NaCl, suspended in150 mM CaCl₂ with 10% glycerol, and incubated at room temperature for 20min. The cells were harvested by centrifugation, resuspended in 0.5 mlof CaCl₂ -glycerol, chilled on ice for 3-5 minutes and frozen. Thesuspensions of cells were stored in liquid nitrogen until use.Preservation and storage did not adversely affect the viability orfrequency of transformation by covalently closed circular DNA. The cellswere thawed in an ice bath and mixed in a ratio of 0.1 ml of cells to0.05 ml of DNA (5 μl pPR3 prepared according to the teaching of Example3C and diluted with 45 μl 0.1 X SSC [standard saline citrate]). Thesamples thus prepared were chilled on ice for 20 minutes, heat shockedat 42° C. for 1 minute, chilled on ice for an additional 10 minutes,then diluted with 0.85 ml of L-broth, incubated at 32° C. for 2 hr,spread on L-agar (disclosed in Miller, 1972) with about 1×10⁹ each ofλKH54hλ and λKH54h80 (both of which are disclosed in Backman et al.1977, Science 196:182), and incubated at 32° C. Transformants wereselected for immunity to bacteriophage λKH54hλ and λKH54h80 at 32° C.The recombinants were tested to verify Ap^(r), Tc^(s), λKH54hλ andλKH54h80 immunity at 32° C., and λKH54hλ and λKH54h80 sensitivity at 42°C. One transformant was selected and designated E. coli K12 C600R_(K)-M_(K) -pPR3. This surviving colony was tested for the expectedphenotypes and used for amplification and isolation of the constructedrecombinant plasmid pPR3. Restriction enzyme analysis of plasmid pPR3showed that the λrex, rather than the λcI, gene was closest to the trpE-insulin B chain gene.

EXAMPLE 5 Amplification and Isolation of Plasmid PR3

The plasmid DNA of E. coli K12 C600R_(K) -M_(K) /pPR3 was amplified withchloramphenicol and isolated by cleared lysate procedure (disclosed inBazaral and Helinski, 1968, J. Mol. Biol. 36:185-194). The covalentlyclosed circular DNA was purified by equilibrium ultracentrifugation inCsCl and propidium di-iodide. The propidium di-iodide was extracted with2-propanol and the DNA was stored in CsCl at -20° C. Working solutionsof DNA were exchanged into SSC/10 buffer (0.015 M NaCl, 0.0015 M sodiumcitrate pH 7.0) by chromatography on Sephadex (PD10*) columns.

EXAMPLE 6 Construction of Plasmid pPR12

A. Isolation of a λcI Gene Containing Linear DNA With PstI and HincIITermini

About 70 μg of plasmid pPR3 (isolated in substantial accordance with theteaching of Example 5), were dissolved in 70 μl 10X PstI buffer (200 mMTris.HCl, pH 7.5, 100 mM MgCl₂, 500 mM (NH₄)₂ SO₄), 50 μl (1 mg./ml.)BSA (bovine serum albumin), and 555 μl H₂ O and then incubated at 65° C.for about 15 minutes. After about 25 μl PstI (1 unit/μl) restrictionenzyme were added, the resultant mixture was incubated at 37° C. forabout 4.5 hours. The resultant PstI restriction fragments were thenconventionally isolated by AGE. Since there are two PstI restrictionsites in plasmid pPR3, a complete PstI digestion results in a ˜3.6 kbfragment and also a ˜4.2 kb fragment. The latter fragment contains thebacteriophage λcI gene of interest. Therefore, the ˜4.2 kb fragment wasrecovered as described herein above. The resultant DNA pellet, whichcomprises the desired ˜4.2 kb restriction fragment was suspended in 50μl TE buffer. The DNA suspension was then incubated at 65° C. for 15minutes and then stored at 4° C. for future use.

About 50 μl of the ˜4.2 kb PstI restriction fragment (prepared above),10 μl 10X HincII buffer (100 mM Tris.HCl, pH 7.9, 600 mM NaCl, 66 mMMgCl₂, 10 mM dithiothreitol), 38 μl H₂ O, and 2 μl (10 units/μl) HincIIrestriction enzyme were incubated at 37° C. for about 20 minutes andthen at 65° C. for about 5 minutes. After the mixture was cooled toabout 20° C. and after about 200 μl TBE were added, the restrictionfragments were conventionally isolated by AGE. The desired ˜0.9 kbPstI-HincII restriction fragment, which contained the bacteriophage λcIgene, was dissolved in 10 μl TE buffer and stored at 4° C.

B. Isolation of a Replicon and tc^(r) Gene Containing Linear DNA WithHincII and PstI Termini

About 100 μl (3.2 μg) plasmid pBR322, 15 μl 10X HincII buffer, 34 μl H₂O, and 1 μl (10 units/μl) of HincII restriction enzyme were incubated at37° C. for about 20 minutes and then at 65° C. for about 5 minutes. Thereaction mixture was then cooled to 4° C. and ethanol precipitated astaught in Example 3. The desired partial HincII digested pBR322 wassuspended in a solution comprising 10 μl 10X PstI buffer and 79 μl H₂ Oand the resultant suspension was then incubated at 65° C. for 5 minutesfollowed by cooling to 4° C. Next, 10 μl (1 mg/ml) BSA and 2 μl PstI (10units/ml) restriction enzyme were added. The resultant reaction mixturewas incubated at 37° C. for 1 hour, then at 65° C. for 5 minutes, andfinally cooled to 4° C. The thus prepared HincII-PstI restrictionfragment was conventionally isolated by AGE. The DNA was dissolved in TEbuffer and stored at 4° C. for future use.

C. Ligation of the Fragment With the λcI Gene and the Linear pBR322 DNAFragment Having PstI and HincII Termini

About 10 μl (1.8 μg) ˜0.9 kb HincII-PstI fragment (prepared in Example6B), and 10 μl (0.9 μg) ˜4 kb PstI-HincII fragment (prepared in Example6A), were mixed and ethanol precipitated twice. The resultant DNA wasdissolved in a solution of 2 μl 5X ligation buffer (250 mM Tris HCl, pH7.8, 50 mM MgCl₂, 25 mM dithiothreitol, and 25% glycerol) and 4.67 μl H₂O. After incubating the solution at 65° C. for 10 minutes, about 3 μl0.66 mM ATP and 0.33 μl (2 units/μl) T4 DNA ligase were added. Theresultant ligation mixture was reacted at ambient temperature for 1.5hours to produce the desired plasmid pPR12. The thus prepared plasmidpPR12 DNA was stored at 4° C. for future use.

EXAMPLE 7 Transformation of Plasmid pPR12 Into E. coli K12 C600R_(k)-M_(k) -

The desired transformation was carried out in substantial accordancewith the teaching of Example 4 except that plasmid pPR12, rather thanplasmid pPR3, was used. Transformants were selected for immunity tobacteriophage λKH54hλ and λKH54h80 at 32° C. The recombinants weretested to verify Ap^(s), Tc^(r), λKH54hλ and λKH54h80 immunity at 32°C., and λKH54hλ and λKH54h80 sensitivity at 42° C. One transformant wasselected and designated E. coli K12 C600R_(k) -M_(k) -/pPR12. Thissurviving colony was tested for the expected phenotypes and used foramplification and isolation of the plasmid pPR12.

EXAMPLE 8 Amplification and Isolation of Plasmid pPR12

The desired amplification and isolation of plasmid pPR12 was carried outin substantial accordance with the teaching of Example 5.

EXAMPLE 9 Construction of Plasmid pPR17

A. Isolation of the ˜4.7 kb EcoRI-BamHI Linear Fragment of Plasmid pPR12Containing the λcI Gene and the Replicon

About 150 μl (20 μg) of plasmid pPR12 DNA (prepared in Example 8), 20 μl10X BamHI buffer (200 mM Tris.HCl, pH 7.0, 1M NaCl, 70 mM MgCl₂, 20 mM2-mercaptoethanol), 2 μl (20 units/μl) BamHI restriction enzyme, and 28μl H₂ O were incubated at 37° C. for 30 minutes, then at ambienttemperature for 1.3 hours, and finally at 65° C. for 5 minutes. Afterthe reaction mixture was cooled to 4° C., about 4 μl (10 units/μl) ofEcoRI restriction enzyme were added. The resultant mixture was thenincubated at 37° C. for 1 hour thus producing the desired ˜4.7 kbrestriction fragment. After conventional isolation by AGE, the desiredDNA pellet was suspended in TE buffer and then stored at -4° C. forfuture use.

B. Isolation of the ˜1.3 kb EcoRI-BamHI Linear Fragment of PlasmidpIA7Δ4Δ1 Containing the Gene for a Fusion Polypeptide of trp 1' E' and AChain of Human Insulin

The desired isolation was carried out in substantial accordance with theteaching of Example 9A except that plasmid pIA7Δ4Δ1, rather than plasmidpPR12, was used. In addition, the EcoRI restriction enzyme was reactedfor only about 1/2 hour since only a partial EcoRI digestion wasdesired. After conventional isolation by AGE, the desired ˜1.3 kbEcoRI-BamHI restriction fragment was used immediately in the ligationprocedure disclosed below.

C. Ligation of the Insulin Fused Gene and the Linear pPR12 DNA FragmentHaving EcoRI and BamHl Termini

About 1.5 μg of the ˜4.7 kb DNA containing solution of Example 9A and1.5 μg of the ˜1.3 kb DNA containing mixture of Example 9B were mixedand ethanol precipitated twice. The pellet was dissolved in a solutioncomprising 6 μl H₂ O and 2 μl 5× ligation buffer and then incubated at65° C. for 10 minutes. After the incubation, the mixture was cooled to15° C. and then about 2 μl 0.66 mM ATP and 0.1 μl (1 unit/μl) T4 DNAligase were added. The ligation reaction was carried out at 15° C. forabout 18 hours producing the desired plasmid pPR17.

EXAMPLE 10 Transformation of Plasmid pPR17 Into E. coli K12 C600R_(k)-M_(k) -

The desired transformation was carried out in substantial accordancewith the teaching of Example 4 except that plasmid pPR17, rather thanplasmid pPR3, was used. Transformants were selected for immunity tobacteriophage λKH54hλ and λKH54h80 at 32° C. The recombinants weretested to verify Ap^(s), Tc^(r), λKH54hλ and λKH54h80 immunity at 32°C., and λKH54hλ and λKH54h80 sensitivity at 42° C. One transformant wasselected and designated E. coli K12 C600R_(k) -M_(k) -/pPR17. Thissurviving colony was tested for the expected phenotypes and used foramplification and isolation of the plasmid pPR17.

EXAMPLE 11 Amplification and Isolation of Plasmid pPR17

The desired amplification and isolation of plasmid pPR17 was carried outin substantial accordance with the teaching of Example 5.

EXAMPLE 12 Construction of Plasmid pPR18

The desired construction was carried out in substantial accordance withthe teaching of Example 9A-C that plasmid pIB7Δ4Δ1, rather than plasmidpIA7Δ4Δ1, was used to generate the ˜1.3 kb EcoRI-BamHI restrictionfragment.

EXAMPLE 13 Transformation of Plasmid pPR18 Into E. coli K12 C600R_(k)-M_(k) -

The desired transformation was carried out in substantial accordancewith the teaching of Example 4 except that plasmid pPR18, rather thanplasmid pPR3, was used. Transformants were selected for immunity tobacteriophage λKH54hλ and λKH54h80 at 32° C. The recombinants weretested to verify Ap^(s), Tc^(r), λKH54hλ and λKH54h80 immunity at 32°C., and λKH54hλ and λKH54h80 sensitivity at 42° C. One transformant wasselected and designated E coli K12 C600R_(k) -M-/pPR18. This survivingcolony was tested for the expected phenotypes and used for amplificationand isolation of the plasmid pPR18.

EXAMPLE 14 Transformation of Plasmid pPR17 Into E. coli K12 294

Plasmids of the present invention are modified against the K-restrictionsystem by transformation into E. coli K12 294. E. coli K12 294 is R_(k)-M_(k) ⁺ so therefore, upon transformation, unmodified plasmid DNAbecomes modified and resistant to restriction by strains with R_(k) ⁺M_(k) ⁺ specificity. Thus E. coli K12 294 transformants are used foramplifying and isolating plasmids of the present invention forsubsequent transformation into R_(k) ⁺ M_(k) ⁺ E coli strains. Suchstrains include, for example, E. coli K12 RV308.

The desired transformation was carried out in substantial accordancewith the teaching of Example 4 except that E. coli K12 294, rather thanE. coli K12 C600R_(k) -M_(k) -, and plasmid pPR17, rather than pPR3,were used. Transformants were selected for Tc^(r). The recombinants weretested to verify Ap^(s), Tc^(r), immunity at 32° C. to λKH54hλ andλKH54h80 and sensitivity at 42° C. to λKH54hλ and λKH54h80. Thetransformants exhibited 100% genetic linkage of the putative plasmidborne markers. One transformant was selected and designated E. coli K12294/pPR17. This colony was tested to verify the expected phenotypes andused for amplification and isolation of plasmid pPR17.

EXAMPLE 15 Amplification and Isolation of Plasmid pPR17

The desired amplification and isolation of plasmid pPR17 was carried outin substantial accordance with the teaching of Example 5 except that E.coli K12 294/pPR17 was used.

EXAMPLE 16 Transformation of Plasmid pPR18 Into E. coli K12 294

The desired transformation was carried out in substantial accordancewith the teaching of Example 14 except that plasmid pPR18, rather thanplasmid pPR17,

EXAMPLE 17 Amplification and Isolation of Plasmid pPR18

The desired amplification and isolation of plasmid pPR18 was carried outin substantial accordance with the teaching of Example 5 except that E.coli K12 294/pPR18 was used.

EXAMPLE 18 Transformation of Plasmid pPR17 Into E. coli K12 RV308

The desired transformation was carried out in substantial accordancewith the teaching of Example 14 except that plasmid pPR17 from Example15 and E. coli K12 RV308 were used.

EXAMPLE 19 Transformation of Plasmid pPR18 Into E. coli K12 RV308

The desired transformation was carried out in substantial accordancewith the teaching of Example 14 except plasmid pPR18 from Example 17 andE. coli K12 RV308 were used.

EXAMPLE 20 Construction of E. coli K12 RV308λcI90/pPR17 byLysogenization with λcI90

E. coli K12 RV308/pPR17 (prepared according to the teaching of Example18) was grown at 32° C. until 35 Klett units and was then transferred to45° C. for 60 minutes. The cells were infected with λcI90 at an moe of20 and incubated at 45° C. for 40 minutes. Colonies were grown at 32° C.on L-agar containing 10 μg/ml tetracycline. The resulting E. coli K12RV308λcI90/pPR17 colonies were tested to verify growth at 32° C. andsensitivity at 42° C.

EXAMPLE 21 Construction of E. coli K12 RV308λcI90/pPR18

The desired construction was prepared in substantial accordance with theteaching of Example 20 except that E. coli K12 RV308/pPR18 (prepared inExample 19), rather than E. coli K12 RV308/pPR17, was used.

EXAMPLE 22 Construction of E. coli K12 C600R_(k) -M_(k) -λcI90/pPR12

The desired construction was prepared in substantial accordance with theteaching of Example 20 except that E. coli K12 C600R_(k) -M_(k) -/pPR12(prepared in Example 7), rather than E. coli K12 RV308/pPR17, was used.

Other representative strains which are constructed in accordance withthe foregoing teaching include:

    ______________________________________                                        Example No.  Name                                                             ______________________________________                                        23           E. coli K12 C600/pPR17                                           24           E. coli K12 C600/pPR18                                           25           E. coli K12 RV308/pPR12                                          26           E. coli K12 RV308 --λcI90/pPR12                           27           E. coli K12 C600 --λcI90/pPR17                            28           E. coli K12 C600 --λcI90/pPR18                            29           E. coli K12 294 --λcI90/pPR17                             30           E. coli K12 294 --λcI90/pPR18                             31           E. coli K12 C600R.sub.k -M.sub.k - --λcI90/pPR17          32           E. coli K12 C600R.sub.k -M.sub.k - --λcI90/pPR18          ______________________________________                                    

EXAMPLE 33 Construction of Plasmid pPR19

The desired construction was made in substantial accordance with theteaching of Examples 1 and 9 except that in Example 1I, a humanproinsulin gene (depicted in FIG. 10) was substituted for the humaninsulin A chain gene resulting in the construction of the proinsulinplasmid pHI7Δ4Δ1 rather than the insulin A chain plasmid pIA7Δ4Δ1 and inExample 9, the ˜1.6 kb EcoRI-BamHI trp 1' E' human proinsulin fusionpolypeptide-encoding fragment of pHI7Δ4Δ1 was substituted for the ˜1.3kb EcoRI-BamHI trp 1' E' human insulin A chain fusionpolypeptide-encoding fragment of pIA7Δ4Δ1. The above-describedconstruction results in the desired ˜6.3 kb plasmid pPR19.

The human proinsulin gene of FIG. 10 can be constructed synthetically bythe modified phosphotriester method (Itakura et al., 1977 and Crea etal., 1978). The gene sequence can also be deduced from either the knownamino acid sequence of human proinsulin or the natural coding sequence(disclosed in Bell et al., 1979, Nature 282:525 and also in Sures etal., 1980, Science 208:57), in view of the degenerate genetic code. Inaddition, the specific nucleotide fragments which are used to synthesizethe EcoRI-sticky end and the portion of the gene that encodes the firstthirty-two amino acids of proinsulin are disclosed in the previouslycited Crea et al., 1978. The remaining portion of the gene can besynthesized or obtained by isolating and transcribing m-RNA transcriptsinto cDNA in accordance with the conventional methods of Goodman, 1979,Methods in Enzymology 68:75, Ullrich et al., 1977, Science 196:1313 andWickens, et al., 1978, J. Biol. Chem. 253:2483. A restriction site andfunctional map of plasmid pPR19 is presented in FIG. 11 of theaccompanying drawings.

EXAMPLE 34 Transformation of Plasmid pPR19 Into E. coli K12 C600R_(k)-M_(k) -

The desired transformation was carried out in substantial accordancewith the teaching of Example 4 except that plasmid pPR19, rather thanplasmid pPR3, was used. Transformants were selected for immunity tobacteriophage λKH54hλ and λKH54h80 at 32° C. The recombinants weretested to verify Ap^(s), Tc^(r), λKH54hλ and λKH54h80 immunity at 32°C., and λKH54hλ and λKH54h80 sensitivity at 42° C. One transformant wasselected and designated E. coli K12 C600R_(k) -M_(k) -/pPR19. Thissurviving colony was tested for the expected phenotypes and used foramplification and isolation of the plasmid pPR19.

EXAMPLE 35 Amplification and Isolation of Plasmid pPR19

The desired amplification and isolation of plasmid pPR19 was carried outin substantial accordance with the teaching of Example 5.

EXAMPLE 36 Transformation of Plasmid pPR19 Into E coli K12 294

The desired transformation was carried out in substantial accordancewith the teaching of Example 14 except that plasmid pPR19, rather thanpPR17, was used. Transformants were selected for Tc^(r). Therecombinants were tested to verify Ap^(s), Tc^(r), immunity at 32° C. toλKH54hλ and λKH54h80 and sensitivity at 42° C. to λKH54hλ and λKH54h80.The transformants exhibited 100% genetic linkage of the putative plasmidborne markers. One transformant was selected and designated E. coli K12294/pPR19 This colony was tested to verify the expected phenotypes andused for amplification and isolation of plasmid pPR19 in substantialaccordance with the teaching of Example 5.

EXAMPLE 37 Transformation of Plasmid pPR19 Into E. coli K12 RV308

The desired transformation was carried out in substantial accordancewith the teaching of Example 14 except that plasmid pPR19, as modified,amplified and isolated in ExampIe 36, and E. coli K12 RV308 were used.

EXAMPLE 38 Construction of E. coli K12 RV308λcI90/pPR19 byLysogenization with λcI90

E. coli K12 RV308/pPR19 (prepared according to the teaching of Example37) was grown at 32° C. until 35 Klett units and was then transferred to45° C. for 60 minutes. The cells were infected with λcI90 at an moe of20 and incubated at 45° C. for 40 minutes. Colonies were grown at 32° C.on L-agar containing 10 μg/ml tetracycline. The resulting E. coli K12RV308λc190/pPR19 colonies were tested to verify growth at 32° C. andsensitivity at 42° C.

Other representative strains which are constructed in accordance withthe foregoing teaching include:

    ______________________________________                                        Example No.  Name                                                             ______________________________________                                        39           E. coli K12 C600/pPR19                                           40           E. coli K12 C600 --λcI90/pPR19                            41           E. coli K12 294 --λcI90/pPR19                             42           E. coli K12 C600R.sub.k -M.sub.k - --λcI90/pPR19          ______________________________________                                    

Method For Determining Stabilities of Host Cells Containing RecombinantPlasmids With and Without Selection

The Tc^(r) gene on the recombinant plasmids was employed to assay thefrequency of cells containing the plasmids. Serial dilutions of culturewere spread on L-agar and grown at 32° C. with and without 10 μg/ml oftetracycline. The frequency of plasmid⁺ cells was taken as the ratio oftetracycline resistant colonies to the total number of colonies thatgrew on L-agar without tetracycline. Alternatively, the colonies onL-agar were replica plated to L-agar with 10 μg/ml of tetracycline andgrown at 32° C. The frequency of plasmid⁺ cells was taken as the ratioof tetracycline resistant colonies to the total number of colonies thatgrew on L-agar without tetracycline. The results are presented aspercentages in Table 2 for strains E. coli K12 RV308/pIA7Δ4Δ1 and E.coli K12 RV308λcI90/pPR17, in Table 3 for strains E. coli K12RV308/pIB7AΔ4Δ1 and E. coli K12 RV308λcI90/pPR18, and in Table 4 for E.coli K12 C600R_(k) -M_(k) -/pPR12 and E. coli K12 C600R_(k) -M_(k)-λcI90/pPR12.

                  TABLE 2                                                         ______________________________________                                        Stabilities of Recombinant Plasmids                                           Number of                                                                             Percentage of Plasmid Retention                                       Culture E. coli          E. coli                                              Doublings                                                                             K12 RV308/pIA7Δ4Δ1                                                                 K12 RV308 --λcI90/pPR17                       ______________________________________                                         9      96               100                                                  30      95               100                                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Stabilities of Recombinant Plasmids                                           Number of                                                                             Percentage of Plasmid Retention                                       Culture E. coli          E. coli                                              Doublings                                                                             K12 RV308/pIB7Δ4Δ1                                                                 K12 RV308 --λcI90/pPR18                       ______________________________________                                         9      96               100                                                  30      79               100                                                  ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Stabilities of Recombinant Plasmids                                           Number of                                                                             Percentage of Plasmid Retention                                       Culture E. coli K12    E. coli K12                                            Doublings                                                                             C600R.sub.k -M.sub.k -/pPR12                                                                 C600R.sub.k -M.sub.k - --λcI90/pPR12            ______________________________________                                        33      87             100                                                    ______________________________________                                    

Results in Tables 2-4 clearly demonstrate the effectiveness of thepresent selective system for maintaining recombinant plasmids inbacterial populations. About 5 percent of the cells in the culture of E.coli K12 RV308/pIA7Δ4Δ1 and about 21 percent of the cells in the cultureE. coli K12 RV308/pIB7Δ4Δ1 were plasmid minus after 30 culturedoublings. None of the cells in the cultures of E. coli K12RV308λcI90/pPR17 and E coli K12 RV308λcI90/pPR18, that had the selectivesystem in place, were plasmid minus. Moreover, the culture of E. coliK12 C600R_(k) -M_(k) -λcI90/pPR12 also showed excellent plasmidstability. Thus, 13% of the cells in the culture of E. coli K12C600R_(k) -M_(k) -/pPR12 were plasmid minus after 33 culture doublings,while all of the cells in the culture of E. coli K12 C600R_(k) -M_(k)-λcI90/pPR12, that had the selective system in place, were plasmid plus.

None of the plasmids of the present invention showed any plasmidsegregation. Thus, the present improved plasmids are furtherdistinguished over those lacking the improvement by the absence ofrecombination with the prophage. In fact, not one plasmid minus colonyhas been observed after growth with the improved selective system inplace.

We claim:
 1. In the method for stabilizing and selecting host cellscontaining recombinant DNA in which host cells are transformed with arecombinant DNA cloning vector which contains both the ˜2.5 kb Bg1 II cIrepressor-containing restriction fragment of bacteriophage λ and a genewhich expresses a functional polypeptide, and in which the transformedhost cells are lysogenized with a lysogenic organism containing a markerwhich is lethal or conditionally lethal in the host cells but which isrepressed in the transformed host cell by the repressor gene containedin the recombinant DNA cloning vector, an improvement wherein theimprovement comprises: transforming the host cells with a recombinantDNA cloning vector comprising(a) the PstI-HincII cI repressor containingrestriction fragment of bacteriophage λ; and (b) a gene which expresseshuman proinsulin; subject to the limitation that the host cells are E.coli, the lysogenic organism is bacteriophage λ and the recombinant DNAcloning vector contains a replicon and a promoter which are notsensitive to the repressor, and subject to the further limitation, thatwhen the transformed host cells are lysogenized with a lysogenicorganism containing a gene which is conditionally lethal, the resultinghost cells are cultured under restrictive conditions.
 2. The method ofclaim 1 wherein the recombinant DNA cloning vector is a plasmid.
 3. Themethod of claim 2 in which the plasmid is pPR19.
 4. A recombinant DNAcloning vector comprising(a) the Pst I- Hinc II c I repressor containingrestriction fragment of bacteriophage λ, (b) a gene, which under thecontrol of a promoter, expresses human proinsulin in E. coli, and (c) areplicon which is functional in E. coli, subject to the limitation thatsaid promoter and said replicon are not sensitive to said repressor. 5.The recombinant DNA cloning vector of claim 4 which is a plasmid.
 6. Therecombinant DNA cloning vector of claim 5 which is plasmid pPR19.
 7. Atransformed host cell selected from the group consisting of E. coli K12C600R_(k) ⁻ M_(k) ⁻ /pPR19, E. coli K12 294/pPR19, E. coli K12RV308/pPR19, E. coli K12 RV308λcI90/pPR19, E. coli K12 C600/pPR19, E.coli K12 C600λcI90/pPR19, E. coli K12 294λcI90/pPR19 and E. coli K12C600R_(k) ⁻ M_(k) ⁻ λcI90/pPR19.
 8. The transformed host cell of claim 7which is E coli K12 RV308λcI90/pPR19.
 9. The transformed host cell ofclaim 7 which is E. coli K12 C600λcI90/pPR19.
 10. The transformed hostcell of claim 7 which is E. coli K12 294λcI90/pPR19.
 11. The transformedhost cell of claim 7 which is E. coli K12 C600R_(k) ⁻ M_(k) ⁻λcI90/pPR19.
 12. A lysogenized transformed E. coli comprising(1) arecombinant DNA cloning vector, said vector comprising(a) the Pst I-HincII c I repressor containing restriction fragment of bacteriophage λ, (b)a gene, which under the control of a promoter, expresses humanproinsulin in E. coli, and (c) a replicon which is functional in E.coli, and (2) a lysogenic organism containing a marker which is lethalor conditionally lethal in E. coli, but which is repressed in thetransformed E. coli by the repressor in said vector, subject to thelimitation that said promoter and said replicon are not sensitive tosaid repressor.